The Toll-like receptor (TLR) superfamily plays a central role in the recognition of invading pathogens and the initiation of an immune response. Ten human TLRs have been identified to date. Each recognises a distinct pathogen-associated molecular pattern (PAMP) leading to the activation of a signalling cascade, which in turn activates the transcription factor NF-κB and also the mitogen-activated protein kinases (MAPKs), p38, c-jun, N terminal kinase (JNK) and p42/44 (reviewed in ref 1 and 2). TLR3 and TLR4 also activate another pathway culminating in the activation of the transcription factor, IFN-regulated factor-3 (IRF3), which binds to the interferon-sensitive response element (ISRE), inducing a subset of genes including IFN-β (3). The TLRs are members of a larger superfamily, called the interleukin-1 receptor (IL-1R)/TLR superfamily, that also contains the IL-1R1 subgroup and the TIR domain-containing adaptor subgroup. All three subgroups possess a cytoplasmic Toll/IL-1 receptor (TIR) domain, which is essential for signalling. The TLRs possess extracellular leucine rich repeats, while the IL-1R1 subgroup have extracellular immunoglobin domains. The adaptor molecules are cytoplasmic and contain no extracellular region.
As mentioned above, each TLR recognises a different PAMP. The first TLR to be discovered was TLR4 and it is essential for the recognition of gram-negative bacterial lipopolysaccharide (LPS) (4, 5). TLR2 coupled with TLRs 1 and 6 recognises diacyl- and triacyl-lipopetides respectively (6). TLR3 recognises dsRNA (7), TLR5 recognises bacterial flagellin (8) while TLR9 recognises unmethylated CpG motifs (9). Once a TLR has recognised a PAMP it must recruit a TIR domain-containing adaptor to activate the subsequent signalling pathway. The first of these adaptors to be identified was MyD88. It plays a key role in TLR and IL-1R signalling (10, 11, 12) and the resulting signalling cascade has been extensively studied (reviewed in 13). Evidence suggests that it is involved in signalling from all TLRs with the exception of TLR3. MyD88-deficient mice failed to respond to IL-1 stimulation, or stimulation of TLR2, TLR5 and TLR9 (11). In the case of TLR4, activation of NF-κB and MAPK still occurred albeit in a delayed manner. In addition, the induction of dendritic cell maturation and the activation of the transcription factor IRF3 were unaffected in MyD88-deficient mice. This suggested that TLR4 requires more than just MyD88 to fully activate its response and that this response could be divided into two categories, the MyD88-dependent response and the MyD88-independent response. NF-κB and TNF production were not impaired in response to TLR3 suggesting that MyD88 is not involved in TLR3 signalling.
The next adaptor to be identified was Mal (MyD88 adaptor-like), which has also been called TIRAP (TIR domain-containing adaptor protein) (14, 15). It was originally thought that this could be the adaptor that mediated the MyD88-independent response to TLR4 but Mal-deficient mice proved that this was not the case and that Mal and MyD88 work together to activate the MyD88-dependent pathway. Like MyD88-deficient mice, Mal-deficient mice showed a delayed activation of NF-κB and MAPK in response to LPS while the activation of dendritic cell maturation and the transcription factor IRF3 were unaffected (15, 16). Mal-deficient mice respond normally to ligands for TLR5, TLR7, TLR9, IL-1 and IL-18 confirming the belief that MyD88 is the only adaptor required by these receptors. TLR3 signalling is also normal in Mal-deficient mice suggesting that neither Mal nor MyD88 are involved in this pathway. Interestingly, the signalling pathway activated by TLR2 was completely abolished in Mal-deficient mice suggesting that Mal and MyD88 are both required for the activation of this pathway (16).
Trif (TIR domain-containing adaptor inducing interferon-β) was the third adaptor to be discovered (17, 18). It was also called TIR-containing adaptor molecule-1 (TICAM-1). Trif, when over-expressed, activated NF-κB albeit to a much lesser extent than Mal or MyD88 but it was a much stronger activator of IFN-β (17). This suggested that it may be involved in the MyD88-independent pathway and Trif-deficient mice proved this (19). NF-κB activation in response to LPS was almost normal in these mice but when these cells were deficient of Trif and MyD88, the NF-κB response to LPS was totally abolished. In Trif-deficient mice the activation of IRF3 in response to LPS was totally abolished again suggesting that Trif is involved in the MyD88-independent pathway activated by TLR4. The activation of IRF3 by TLR3 was also abolished in Trif-deficient cells and the activation of NF-κB was severely impaired suggesting that Trif is the sole adaptor used by TLR3.
It was discovered that Trif could not bind directly to TLR4 (18) suggesting that a bridging adaptor is needed to bind it to TLR4. That bridging adaptor has now been discovered by several groups and is called TRAM (Trif-related adaptor molecule) (20) or TICAM-2 (TIR-containing adaptor molecule-2) (21) or TIRP (TIR domain-containing protein) (22).
TRAM binds directly to TLR4 but not to the other TLRs (21). Overexpression of TRAM led to a mild induction of IRF3, IRF7 and NF-κB, independent of MyD88. A dominant negative form of TRAM inhibited activation of NF-κB and IRF3 by LPS, but had no effect on the activation of either of these transcription factors by the TLR3 ligand, Poly(I:C). Overexpression of TRAM, along with Trif, lead to the translocation of IRF3 to the nucleus (20). A dominant negative form of Trif largely suppressed the ability of TRAM to activate NF-κB and IFN-β while MyD88 and Mal dominant negative mutants had no effect.
TRAM cannot function in Trif-knockdown RAW cells, suggesting that TRAM is working upstream of Trif on the TLR4 pathway. The generation of TRAM-deficient mice (23) added weight to this theory. These mice showed that TRAM was essential for activation of the MyD88-independent pathway in response to TLR4 and that it was not involved in other TLR pathways.
The inventors have now surprisingly found that the adapter molecule TRAM is rapidly phosphorylated by protein kinase C epsilon following the binding of LPS to the TLR4 receptor (Toll Like Receptor 4). It is defined that TRAM is phosphorylated by protein kinase C epsilon at the site of the serine 16 residue. Assays directed to monitoring the phosphorylation of TRAM may be a useful tool in determining the activation of TLR4 and in particular whether LPS signalling through the TLR4 receptor is functioning properly in different environments.